JP7227159B2 - Systems and methods for the production of recombinant IL-11 in yeast - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US Provisional Patent Application No. 62/446,762, filed January 16, 2017. These and all other referenced external materials are hereby incorporated by reference in their entirety. If the definition or usage of a term incorporated herein by reference conflicts or is contrary to the definition of that term provided herein, the definition of that term as provided herein shall control. It is regarded.

TECHNICAL FIELD The field of the invention is the production and subsequent purification of recombinant IL-11, especially in yeast.

The background description includes information that may be useful in understanding the present invention. It is not intended that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. It is not allowed.

Interleukin IL-11 has considerable therapeutic potential, but production of IL-11 in suitable scale and purity has proven difficult. Expression of recombinant IL-11 in bacteria has been attempted due to its lack of glycosylation. However, the resulting protein tends to be expressed as insoluble inclusion bodies, resulting in poor yields. This is probably due to improper folding. Attempts have been made to express IL-11 in yeast, but to date such methods have exhibited low yields and have required the use of toxic organic solvents.

One approach to address this is to express recombinant IL-11 as a fusion protein with more desirable expression characteristics. Commercially available IL-11 is typically isolated from a fusion protein expressed in E. coli. Unfortunately, the use of enterokinase to generate IL-11 fragments from fusion proteins results in product heterogeneity. Similarly, US Patent Application Publication No. 2009/0010872 (Mackiewicz) discloses recombinant IL-11 fusion proteins incorporating both IL-11 and soluble IL-11 receptor sequences, and have described the expression of such fusion proteins in insect or mammalian cells. However, recovery of IL-11 from such fusion proteins requires additional processing steps that can cleave the fusion protein and result in variations in length and/or sequence of the product IL-11 fragments. Moreover, such cells have complex culture requirements that can complicate downstream purification of the desired product.

For example, US Patent Application Publication No. 2007/0275889 describes the use of plasmids encoding both IL-11 sequences and chaperonins, and expression of such plasmids in insect or mammalian cells in culture. are doing. Chaperonins provide proper folding and help prevent aggregation of expressed IL-11. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the definition or use of a term in an incorporated reference contradicts or runs contrary to the definition of that term provided herein, the definition of that term provided herein shall apply; The definition of that term in reference does not apply. However, as noted above, such cell culture conditions can complicate subsequent purification steps. Moreover, expression in mammalian and insect cells in culture is generally much lower than that of bacteria or yeast.

U.S. Patent Application Publication No. 2009/0010872 U.S. Patent Application Publication No. 2007/0275889

Therefore, there remains a need for simple, effective and scalable methods for providing substantially pure and active IL-11.

In some embodiments, numbers expressing amounts of ingredients, properties such as concentrations, reaction conditions, etc. used to describe and claim certain embodiments of the present invention are sometimes referred to as the term “about” should be understood to be modified by Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, numeric parameters should be interpreted by applying normal rounding techniques, taking into account reported significant digits. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention can contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

As used throughout the specification and claims, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "middle" includes "middle" and "above" unless the context clearly dictates otherwise.

Unless the context indicates to the contrary, all ranges recited herein should be interpreted as inclusive of their endpoints, and open-ended ranges should be interpreted as inclusive only of commercially practical values. is. Similarly, all value lists should be considered inclusive unless the context indicates otherwise.

Recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise specified herein, each individual value having a range is incorporated herein as if it were an individual listing herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples, or use of exemplary language (e.g., "like") provided herein with respect to particular embodiments, are merely to better clarify the invention and may otherwise be It does not limit the scope of the claimed invention. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included or removed from a group for reasons of convenience and/or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the group as modified and thus satisfy all written descriptions of Markush groups used in the appended claims.

SUMMARY OF THE INVENTION The present subject matter provides devices, systems and methods that provide highly purified recombinant IL-11 with reduced dimer and oxide content compared to prior art methods.

One embodiment of the inventive concept is a method of producing IL-11 comprising introducing into yeast an expression vector encoding recombinant IL-11, wherein the encoded recombinant IL-11 is a fusion protein. not the form. Yeast is cultured in medium under conditions that induce expression of IL-11, and the supernatant is then separated from the medium solids. This supernatant is then treated with a sufficient amount of polyethylene glycol to form a suspension containing the precipitate. For example, polyethylene glycol can be provided at a final concentration of about 4% (w/v) to about 12% (w/v) and/or about 6% (w/v) to about 9% (w/v). . Such polyethylene glycols can have molecular weights ranging from about 2,000D to about 20,000D, and/or from about 4,000D to about 12,000D.

This precipitate is solubilized in a solution containing a denaturant to produce a crude IL-11 solution. Suitable denaturants include urea, guanidine hydrochloride, and/or detergents such as dodecyl sulfate and/or N-sarkosyl. For example, the concentration of guanidine hydrochloride can be from about 4M to about 10M or from about 5M to about 9M in such solubilization steps. The denaturant concentration is then reduced (eg, the guanidine hydrochloride concentration can be reduced to 0.7 M or less) to produce a refolded IL-11 solution. In some embodiments, the step of reducing the concentration of the denaturant comprises incubating at 18° C.-25° C. for about 1 hour after reducing the concentration of the denaturant. Denaturant concentration can be reduced by any suitable means, including dilution and/or buffer exchange. Refolding of IL-11 can be performed at protein concentrations from about 0.1 mg/mL to about 10 mg/mL, and/or less than about 2 mg/mL, and can be performed without co-solutes. Refolding can be performed at a pH of about 4 to about 12, or a pH of about 7 to about 11. The refolded IL-11 solution is then contacted with an ion exchange medium, followed by elution of the purified IL-11 from the ion exchange medium (eg, cation exchange medium).

In some embodiments, the above methods include additional processing steps. In some embodiments, purified IL-11 is contacted with a hydrophobic interaction medium. Suitable hydrophobic interaction media include butyl, hexyl, octyl, and/or phenyl media. Polished IL-11, which has a reduced content of oxidized IL-11 compared to purified IL-11, is then eluted from the hydrophobic interaction medium. The purified IL-11 obtained can have a purity of at least 95%, for example containing no more than about 5% oxidized IL-11 and/or no more than 1% dimers of IL-11. . Polished IL-11 is typically about 4×10 ⁶ U/mg to about 1.2×10 ⁷ U/mg (e.g., about 6×10 ⁶ U/mg) when tested with the 7TD1 cell line. /mg) of biological activity.

Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, in which like reference numerals denote like components.

Results of high-density fermentation of rhIL-11 expression are shown. FIG. 1A shows growth curves over different time points. Media were harvested at different time points after induction and analyzed by non-reducing SDS-PAGE and immunoblotting. FIG. 1B shows typical results of non-reducing SDS-PAGE with Coomassie Blue staining. FIG. 1C shows typical results from Western immunoblotting. M represents a protein marker. Results of high-density fermentation of rhIL-11 expression are shown. FIG. 1A shows growth curves over different time points. Media were harvested at different time points after induction and analyzed by non-reducing SDS-PAGE and immunoblotting. FIG. 1B shows typical results of non-reducing SDS-PAGE with Coomassie blue staining. FIG. 1C shows typical results from Western immunoblotting. M represents a protein marker. Results of high-density fermentation of rhIL-11 expression are shown. FIG. 1A shows growth curves over different time points. Media were harvested at different time points after induction and analyzed by non-reducing SDS-PAGE and immunoblotting. FIG. 1B shows typical results of non-reducing SDS-PAGE with Coomassie blue staining. FIG. 1C shows typical results from Western immunoblotting. M represents a protein marker. A representative non-reducing 16% SDS-PAGE gel illustrating recovery of rhIL-11 after aqueous two-phase extraction is shown. FIG. 4 provides a far-UV CD spectrum of crude rhIL-11 isolated from expression medium using liquid two-phase extraction. Size exclusion chromatography results of rhIL-11 precipitated from fermentation medium (lower panel) and rhIL-11 reference standard results (upper panel) are shown. Shown are the results of isolation of rhIL-11 from fermentation medium containing 0.1% Tween-80 by liquid chromatography using a Capto-S column (cation exchanger). The red line represents in-line conductivity and the blue line represents UV absorbance at 280 nm. Shaded areas represent fractions judged to be suitable for pooling. Shows the results of isolation of rhIL-11 on Capto-S cation exchangers after refolding in the presence of denaturants. FIG. 6(A) shows the results from using 8M urea. FIG. 6(B) shows results from the use of 6M guanidine hydrochloride. In both figures, the red line represents in-line conductivity and the blue line represents UV absorbance at 280 nm. Arrows indicate the elution position of renatured rhIL-11. Refolding yields by dissolving various concentrations of precipitated proteins in the presence of 7M GdHCl. Results of isolation of refolded rhIL-11 in the presence or absence of co-solutes are shown. The red line represents in-line conductivity and the blue line represents UV absorbance at 280 nm. Refolding yield of rhIL-11 at different pH is shown. Shown are the results of isolation of renatured rhIL-11 by liquid chromatography using a Capto-S column (cation exchanger). The red line represents in-line conductivity, the green line represents pH and the blue line represents UV absorbance at 280 nm. Shaded areas represent fractions judged to be suitable for pooling. 4 shows the results of molecular weight measurement by LC/MS. FIG. 11A shows a typical ion chromatogram. FIG. 11B shows a major peak with a deconvoluted mass of 19,046.7 Da, consistent with the expected molecular weight of 19,047. The small peak observed at the deconvoluted mass of 19,062.5 Da is probably due to oxidized IL-11, as the additional 16 Da can be accounted for by a single oxygen. 4 shows the results of molecular weight measurement by LC/MS. FIG. 11A shows a typical ion chromatogram. FIG. 11B shows a major peak with a deconvoluted mass of 19,046.7 Da, consistent with the expected molecular weight of 19,047. The small peak observed at the deconvoluted mass of 19,062.5 Da is probably due to oxidized IL-11, as the additional 16 Da can be accounted for by a single oxygen. Figure 3 shows the results of size exclusion chromatography of the main peak elution fractions from ion exchange on a Capto-S column. Typical results of polishing rhIL-11 by hydrophobic interaction chromatography using a Butyl HP column to remove oxidized rhIL-11 are shown. The red line represents in-line conductivity and the blue line represents UV absorbance at 280 nm. Shaded areas represent fractions judged to be suitable for pooling. Representative results of a purity study of purified rhIL-11 using non-reducing 16% SDS-PAGE stained with Coomassie Brilliant Blue are shown. Protein samples were loaded at 0.2-5.0 μg. Representative results of a purity study of rhIL-11 and related proteins assayed by RP-UPLC are shown. Oxidized rhIL-11 was present at approximately 2.5% and an unknown impurity was present at approximately 1.6%. Representative results of a purity study of monomeric rhIL-11 as determined by SEC-UPLC are shown. The purity of monomeric rhIL-11 was approximately 99.0%. FIG. 2 provides the mass spectrum m/z of the purified IL-11 product. A deconvoluted mass of 19,045.7 Da was obtained, consistent with the expected molecular weight of 19,047 Da. A typical total ion chromatogram of tryptic peptides derived from hydrolysis of rhIL-11 is shown. The identity of each peptide was confirmed by m/z and MS/MS fragmentation. Representative results of cell proliferation assays performed with purified rhIL-11 are shown.

The following description includes information that may be useful in understanding the present invention. It is not intended that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. It is not allowed.

The present subject matter provides devices, systems and methods by which recombinant human IL-11 can be expressed in yeast and recovered from the culture medium as an active, substantially pure, monomeric protein. Recombinant proteins are precipitated with solvent exclusion reagents (eg, polyethylene glycol), solubilized in the presence of chaotropes or denaturants (eg, guanidinium), and renatured to provide proper protein folding. Chromatographic steps such as ion exchange (eg cation exchange) and/or hydrophobic interaction chromatography (eg using butyl-substituted chromatographic media) can be incorporated into the method of the present concept.

Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings, in which like reference numerals denote like components.

In some embodiments, numbers expressing amounts of ingredients, properties such as concentrations, reaction conditions, etc. used to describe and claim certain embodiments of the present invention are sometimes referred to as the term “about” should be understood to be modified by Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, numeric parameters should be interpreted by applying normal rounding techniques, taking into account reported significant digits. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention can contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

As used throughout this specification and the claims that follow, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. . Also, as used in the description herein, the meaning of "middle" includes "middle" and "above" unless the context clearly dictates otherwise.

Recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise specified herein, each individual value is incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples, or use of exemplary language (e.g., "like") provided herein with respect to particular embodiments, are merely to better clarify the invention and may otherwise be It does not limit the scope of the claimed invention. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included or removed from a group for reasons of convenience and/or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the group as modified and thus satisfy all written descriptions of Markush groups used in the appended claims.

In deriving the methods and compositions of the present concept, the PichiaPink™ Expression System from Invitrogen was used to establish stable rhIL-11 high-level expressing clones secreting rhIL-11. This vector expresses the α-mating factor pre-sequence from Saccharomyces cerevisiae, which is a short signaling peptide that directs the recombinant protein to the extracellular medium. A special feature of the PichiaPink™ Expression System is the use of the ADE2 gene promoter and gene product for selection of high copy number clones. Mutations in ADE2, the gene responsible for de novo biosynthesis of purine nucleotides, lead to the accumulation of purine precursors that impart a red/pink color to transformed colonies. The expression strain is an ade2 auxotroph that cannot grow on media lacking adenine. Transformation of the expression host with the plasmid allows the strain to grow on media lacking adenine, and the short ADE promoter sequence facilitates screening of clones incorporating high copy number. Low copy number colonies appear pink. On the other hand, white colonies are high copy number clones.

Prior art methods of producing recombinant human IL-11 in yeast using Pichia pastoris suffer from low production yields at yeast expression and product purification levels. We found that high cell density fermentation results suggested both expression and low recovery (1-5%) of inactive rhIL-11 after cation exchange purification. ELISA quantification of expression levels in media was found to underestimate IL-11 content (by approximately 90% reduction) when compared to SDS-PAGE analysis against known amounts of reference. The inventors conclude that prior art methods provide low yields due to misfolding and/or aggregation that has been addressed to some extent using reversed-phase chromatography and subsequent removal of organic solvents. attached.

The existence of secreted, soluble but misfolded rhIL-11 has been previously reported, as it is generally believed that misfolded proteins aggregate intracellularly or are salvaged for degradation. No (9). The concept method of the present invention utilizes the expression and purification of recombinant human IL-11 using the Pichia pastoris expression system and its isolation from yeast fermentation medium without the use of reversed-phase chromatography.

Alternative approaches to renaturation of rhIL-11 were explored, including the use of chaotropes/denaturants such as urea, SDS, ammonium sulfate, and guanidine hydrochloride. We found that high concentrations of guanidine hydrochloride can disrupt the interactions that lead to self-aggregation of rhIL-11, allowing proper refolding of monomeric rhIL-11 when the concentration of denaturant is reduced. made it possible to

The method of producing rhIL-11 from culture medium begins with a two-phase extraction to precipitate rhIL-11 from the culture medium. Fermentation, including introduction of salts (e.g., ammonium sulfate, sodium sulfate), organic solvents (e.g., methanol, ethanol, acetone, etc.) and/or hydrophilic polymers (e.g., dextran, dextrin, cyclodextrin, polyethylene glycol/PEG, etc.) Precipitation can be performed by any suitable means that provides selective or partially selective precipitation of rhIL-11 from the medium. For example, a final concentration of 8% (w/v) PEG (eg, PEG-8000) with a molecular weight of 8,000 Da can be used. Proteins precipitated from the fermentation medium are separated from the fermentation medium for further processing. This separation can be accomplished by any suitable means including sedimentation, decantation, filtration, and/or centrifugation. In some embodiments, precipitated proteins can be washed one or more times (eg, using a wash buffer containing a precipitant) prior to further processing.

The precipitated crude protein is then dissolved in a buffer containing denaturants, which can help break up protein aggregates. Suitable denaturants include chaotropic agents (e.g. urea, guanidine salts, isothiocyanate salts, etc.) and surfactants (e.g. ionic detergents such as dodecyl sulfate, Tween-20 and/or Tween-80, etc.). nonionic surfactants, and zwitterionic surfactants). For example, guanidine hydrochloride at a final concentration of 7 M can be used to disrupt protein aggregates and resolubilize precipitated proteins.

However, use of such denaturants inevitably results in denaturation of the desired rhIL-11 product. This denaturation can be reversed to yield renatured/refolded rhIL-11 by removing the denaturant or reducing the concentration of the denaturant. This removal can be rapid or gradual. Removal of denaturants can be accomplished by any suitable means, including dilution (eg, with buffers containing reduced or no denaturants) and buffer exchange. Buffer exchange can be gradual (eg, by dialysis) or relatively rapid (eg, by diafiltration, size exclusion chromatography, etc.). It should be appreciated that methods such as dialysis and diafiltration are relatively ineffective at removing low-CMC surfactants. In some embodiments, direct dilution with detergent-free buffer can reduce the concentration of denaturant sufficiently to allow refolding and renaturation of rhIL-11.

Surprisingly, we have found that guanidine HCl provides both solubilization of precipitated rhIL-11 and subsequent chaotrope removal or reduction upon activity/regeneration to native conformation. It was found to be more effective than chaotropic agents. Although guanidine HCl has been found to be very effective for this purpose, Applicants have found that other chaotropic agents (e.g. urea, isothiocyanate salts, etc.) and/or surfactants have been optimized for their use. We believe that under certain conditions it can be equally effective.

It should be understood that correct refolding or renaturation of denatured rhIL-11 may be a function of factors other than the concentration of denaturant. For example, protein concentration during renaturation can affect the extent to which renaturation occurs and the formation of undesirable by-products such as dimers and higher order aggregates. A high protein concentration during renaturation is desirable from a process efficiency standpoint, but such considerations must be balanced against the yield and purity of the final product. The protein concentration during production of renatured or refolded rhIL-11 in the methods of the invention can range from about 0.1 mg/ml to about 10 mg/ml. Surprisingly, the inventors have found that renaturation or production of refolded rhIL-11 yields optimal results at protein concentrations of less than 2 mg/mL. This can be conveniently accomplished in combination with reducing the concentration of denaturant by diluting with a buffer that does not contain denaturant.

Similarly, pH during the renaturation step can affect refolding or renaturation of denatured rhIL-11. We have found that regeneration can be performed at a pH ranging from about 4 to about 12. In a preferred embodiment, regeneration can be performed at a pH in the range of about 7 to about 11. If necessary, the pH can be adjusted by appropriate addition of acid (such as HCl) or base (such as NaOH) before or during regeneration. Alternatively, the pH can be adjusted by adding a buffering compound (e.g., phosphate, bicarbonate, Tris, HEPES, etc.) to the regeneration solution, or by buffer exchanging the regeneration solution for a buffer solution of appropriate pH. can.

Following renaturation/refolding, rhIL-11 can be subjected to two chromatographic procedures. The first of these is ion exchange using a cation exchanger. Suitable cation exchangers include weak cation exchangers (eg ion exchangers bearing carboxyl groups) and strong cation exchangers (eg ion exchangers containing sulfonic acid groups). Such cation exchange can be performed using ion exchange membranes, ion exchange resins, and/or phase transfer solvents. In a preferred embodiment, ion exchange is performed using a cation exchange resin packed in a chromatography column, which facilitates collection of specific fractions. The rhIL-11 preparation can be applied at low ionic strength, which allows rhIL-11 to associate or bind with the cation exchanger. rhIL after passing unbound contaminants by increasing the ionic strength of the applied buffer (e.g., by increasing the concentration of NaCl or other salts) or by changing the pH of the applied buffer. -11 can be eluted or liberated from the cation exchanger. Elution can be performed in steps or in a gradient. It should be appreciated that applying high sample loads (greater than 5 mg/mL) to some cation exchange columns can reduce losses due to non-specific binding and improve process efficiency. For example, use of a Capto-S strong cation exchange column can allow loading at 13 mg/mL and provide active rhIL-11 with a step recovery of 40%-60%.

In some embodiments, a second chromatography step utilizing hydrophobic interaction chromatography removes additional contaminants and separates the products of cation exchange to provide a more highly purified rhIL-11. Applies to Hydrophobic interaction chromatography can be performed using membranes or resins containing suitable hydrophobic moieties. Suitable hydrophobic moieties include propyl, butyl, hexyl, octyl, and phenyl groups. In some embodiments, salts (such as sulfate or phosphate) can be added to the rhIL-11 solution prior to hydrophobic interaction chromatography. Such salts may also help elute rhIL-11 from the cation exchanger in the previous ion exchange step. In a preferred embodiment, hydrophobic interaction chromatography is performed using a hydrophobic interaction resin packed in a chromatography column, which facilitates collection of specific fractions during elution. rhIL-11 is eluted from such hydrophobic interaction columns by decreasing the ionic strength of the applied buffer and/or increasing the organic solvent and/or detergent concentration of the applied buffer. can be This change in buffer composition can be a change in steps or can be applied as a gradient. It should be understood that rhIL-11 can be applied to such hydrophobic interaction columns at high protein loadings, thereby improving process efficiency. For example, using a hydrophobic interaction chromatography column with a sample load of 6-8 mg/mL, a rhIL-11 product purity of greater than about 95% (due to removal of oxidized rhIL-11 and other impurities) was achieved. , with a process yield of about 50%. The resulting purified rhIL-11 can then be concentrated to at least 6 mg/mL and subjected to buffer exchange with 10 mM sodium phosphate pH 7 buffer for refrigeration. A typical overall yield can be about 20-25%. The final purified bulk from such methods has been characterized for identity, purity and potency. Such characterization indicates that this method can yield potent rhIL-11 in high purity.

We believe that oxidation of rhIL-11 is a significant contributor to product contamination and that chromatography steps are performed to reduce the presence of oxidized rhIL-11 impacts yield. I paid attention to Oxidation of recombinant proteins is due to the presence of reactive oxygen species including superoxide (O ₂ −) and its protonated forms (HOO*), hydrogen peroxide (H ₂ O ₂ ), and other hydroxyls (OH*). , mostly occurring during processing and storage (17). Protein oxidation of sulfur-containing residues plays a particularly important role in stability. Oxidative stress, especially of protein therapeutics, can lead to a variety of medical consequences, such as decreased potency and increased immunogenicity (18). Oxidative damage is often associated with dissolved oxygen during fermentation, which is inevitable in aerobic fermentation. We believe that modifications to the fermentation process can be made to minimize the production of oxidized proteins during culture as well as optimize downstream polishing processes for selective removal of oxidized proteins.

It should be appreciated that rhIL-11 preparations resulting from such methods are of higher biological activity and higher purity compared to rhIL-11 produced by prior art methods. When tested for biological activity using the 7TD1 cell line, the polished rhIL-11 eluted from the hydrophobic interaction column as described above yielded about 4×10 ⁶ U/mg protein to about 1.2× They can have biological activities in the range of 10 ⁷ U/mg protein. It is typically about 6×10 ⁶ U/mg protein. The purity of such rhIL-11 preparations may be greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. Typically rhIL-11 was produced with >95% purity as described above. As noted above, oxidized rhIL-11 is commonly found in products from aerobic fermentation. Preparations of rhIL-11 prepared as described above typically contain 5% or less of oxidized rhIL-11. Similarly, a preparation of rhIL-11 prepared as described above contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% dimeric rhIL-11 11 (ie rhIL-11 dimer) content. Typically, such preparations contain less than 1% dimeric rhIL-11 content.

The following are illustrative examples of the inventive concept and should not be considered limiting.

Materials PichiaPink™ Expression System (#A11152, Al 1154) was obtained from Invitrogen Life Technologies. Restriction enzymes and polymerases for clone construction were purchased from Thermo Fisher Scientific's FastDigest enzymes. Antifoam 204 (#A6426), yeast nitrogen base without amino acids (#Y0626) were obtained from Sigma-Aldrich. The yeast-derived rhIL-11 reference standard was provided by Hangzhou Jiuyuan Gene Engineering Company (Lot#20121005/1006/1007/1008 & 20150402). 8-16% Gradient SDS PAGE (#25268), StartingBlock Blocking Buffers (#37579), Blocker™ Casein (#37583), Transfer Buffer Methanol Free (#35045), TBS Tween 20 Buffer (#28360) and 1-Step Ultra Reagents and materials for immunoblotting were purchased from Thermo scientific, including TMB (#37574), Gibco® 2-mercaptoethanol (#21985-023) and Novex Tris-Glycine 16% polyacrylamide gel (XP00162BOX). Human IL-11 Affinity Purified Polyclonal Goat IgG (#AF-218-NA) and Donkey anti-goat IgG HRP affinity purified polyclonal (#HAF109) were obtained from R&D systems. 7TD1 mouse myeloma cells fused with C57B1/6 splenocytes were obtained from DSMZ (No. ACC23). Sequencing grade trypsin modified from bovine pancreas (catalog number 11418025001) was purchased from Roche Diagnostics. Mouse IL-11 receptor alpha (catalog number MBS553276) was purchased from MyBioSource, Inc.; Obtained from CellTiter96® Aqueous Non-Radioactive Cell Proliferation Assay (MTS) (catalog number G5430) was purchased from Promega. RPMI 1640 (#SH30255.01), HI FBS (H#SH30071.03HI) and Strep/Pen (HYCLONE, USA, SV30010) were purchased from HYCLONE, USA. Purified resins Capto S (product code 17-5316-10), Capto Q (product code 17-5441-01) and Butyl HP (product code 17-5432-01) were obtained from GE Healthcare Life Sciences. Trifluoroacetic acid (catalog number 302031) and acetonitrile (catalog number 34967) for HPLC runs were purchased from Sigma-Aldrich. Polyethylene glycol 8000 (#408050010), DL-methionine (#125652500) and guanidine hydrochloride (#364790025) were obtained from Acros Organic. Ammonium sulfate (#11566) was obtained from Alfa Aesar. Non-animal derived phytone peptone (#210931) was obtained from Becton Dickinson.

Cloning and Cell Banking of rhIL-11 Expressing Yeast Clones A recombinant human IL-11 gene was synthesized and cloned into the pPINKα-HC yeast expression vector containing the gene encoding the α-factor signal peptide, in which the Glu-Ala repeats are missing. had lost The resulting recombinant vector was transformed into a protease-deficient strain by electroporation to generate stable clones. Approximately 40 clones from each transformation were selected, visualized using Coomassie blue staining, and screened for high IL-11 expressing clones by visual inspection of expression intensity on SDS-PAGE (data not shown). Protein identity was confirmed by Western immunoblot using a rhIL-11 specific antibody. Research masters and working cell banks were prepared accordingly and cryopreserved.

To ensure consistent expression in quantity and quality, the selected clone pPINKS2-IL11-24 was passaged for 4 generations to expand the cell master bank followed by the working cell bank after cloning and purification. Master and working cell banks were stored at -80°C for long-term storage.

High Density Fermentation A 1 liter fermentation system (BIOSTAT® B Bioreactor, Sartorius) was used to assess rhIL-11 expression levels of selected clones in high cell density cultures using fed-batch fermentation. Fermentation was started by using a thawed vial from the working cell bank to inoculate 15 mL of MGM (minimal glycerol medium; 0.2 μm filtration) medium lacking adenine. The composition of MGM is as follows.

1.34% Yeast Nitrogen Base without Amino Acids 1% Glycerol 4ppm Biotin

After culturing at 30° C. with shaking at 250 rpm for 20 hours, the resulting medium was used to inoculate 100 mL of BSM (fermentation basal salt medium) containing 0.5% phytone peptone (pH 5) for an additional 48 hours. The composition of BSM was prepared as follows.

Composition of BSM Glycerol (50%) 80 mL
Phosphoric acid (28%) 26.7 mL
0.9 g of calcium sulfate
Magnesium sulfate 14.9g
Potassium hydroxide 4.1g
Potassium sulfate 18.2g
Phytone peptone 5.0g
PTM1 trace salt 4.4 mL
Water was added to bring the final volume to 1 L.

The pH of the broth was adjusted to 5.0 and the filtered PTM1 trace salts were added slowly to prevent precipitation. PTM1 trace salts were prepared as follows.

Composition of PTM1 trace salts Cupric sulfate-5H ₂ O 6.0 g
Sodium iodide 0.08g
Manganese sulfate-H ₂ O 3.0 g
Sodium molybdate-2H ₂ O 0.2 g
Boric acid 0.02g
Cobalt chloride 0.5g
Zinc chloride 20.0g
Ferrous sulfate- _7H2O 65.0 g
Biotin 0.2g
Sulfuric acid 5.0 mL
Water was added to bring the final volume to 1 L.

Then, in a 1 L vessel, 600 mL of Fermentation Basal Salt Medium (BSM) containing 0.5% (w/v) soy phytone peptone was mixed with 500 μL of antifoam, followed by glycerol under the same culture conditions. During the batch phase, freshly cultured seed cells were inoculated. The dissolved oxygen level-pO2 value was maintained at 80% by adjusting the agitation speed and the air inlet flow rate was set at 0.3 L/min. In the glycerol-fed batch phase, 50% glycerol was fed to the vessel at a limiting rate of 6 mL/hr/L to promote growth. Cell density was monitored by measuring OD600nm at different time points until the OD reached approximately 180-200. During this phase, the pO2 value was maintained above 30% by increasing the stirring speed and air inflow. The expression of rhIL-11 was increased for the first 4-5 hours with 30% (v/v) methanol at 5.5 mL/h/L followed by 50% (v/v) at 5.5-9 mL/h/L. It was induced in the methanol-fed batch by feeding methanol. The pO2 value was maintained above 20% by increasing the stirring speed and air inflow. Medium was harvested after 48-72 hours of induction.

Expression Levels of rhIL-11 Expressing Clones The protein content of the medium at the desired expression level was found to be consistently underestimated by ELISA due to the presence of misfolded IL-11 (antibody was found to be almost undetectable by cytotoxicity), determined by visual inspection against the intensity of a reference standard on the same SDS-PAGE gel. Alternatively, protein content can be quantified using densitometry software such as GelQuant.NET (version 1.8.2) provided by BiochemLab Solutions. Accurate quantification of expression levels was also achieved using RP-HPLC. Secreted rhIL-11 in the medium was precipitated by adding 8% (w/v) PEG-8000 and collected after centrifugation. The pellet was resuspended in pH 8 sodium phosphate buffer containing 7M guanidine hydrochloride. After removing insoluble particulates by centrifugation, the expression productivity was calculated by comparing the integrated area to a reference standard of known concentration using the following chromatographic procedure for RP-HPLC operation.

Column: PLRP-S column (Agilent), 8 μm, 2.1×150 mm, 300 A (Angstrom) pore size, equipped with a guard cartridge Mobile phase A: 0.1% (v/v) TFA in water; Mobile phase B: 0.1% (v/v) TFA in 90% (v/v) acetonitrile
Flow rate: 0.2 ml/min Detection: 215 nm
Injection volume: 5-10 μL

Gradients: A typical solvent gradient is shown in Table 1.

Protein Concentration of Purified rhIL-11 The concentration of rhIL-11 after the chromatography step was determined by UV absorbance at 280 nm using a UV/Vis microplate and cuvette spectrophotometer (eg MultiskanGO from ThermoScientific). . The extinction coefficient (Ec) in units of M ⁻¹ cm ⁻¹ at 280 nm measured in water was calculated as 17,990 using the following equation.
Ec = number (Tyr) x 1490 + number (Trp) x 5500 + number (Cys) x 125

Protein concentration in moles is calculated as follows.
concentration = (absorbance at 280 nm)/Ec

Alternatively, protein concentration can be determined directly by UV spectroscopy at 280 nm using an absorbance value of 0.944 for a 0.1% (ie 1 mg/ml) solution. Protein quantitation using absorbance at 280 nm measures the absorbance of aromatic amino acids such as tryptophan and tyrosine and does not detect the presence of PEG moieties. As a result, the protein weight concentrations described herein exclude the presence of PEG molecules. Both values can be calculated by ProtParam, a tool for calculating physical and chemical parameters of a given protein (10).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, staining and immunoblotting Apparent molecular weights of proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Biorad's Mini-PROTEAN system in combination with precast gels. evaluated. The resulting polyacrylamide gels were visualized according to Coomassie blue or silver staining.

Protein immunoblotting was performed using Mini Trans-Blot Cells from Bio-Rad. After protein electrophoresis, proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane using a current of 300 mA for 1 hour. The nitrocellulose membrane was then blocked with blocking buffer for 30 minutes overnight, followed by 6 washes of 5 minutes each with TBS-Tween 20 buffer. Proteins of interest were detected on the membrane by incubating for 1 hour at room temperature with a casein block diluted (1:2,000) primary antibody against human IL-11 (goat IgG). After washing the membrane six times for 4 min each with TBS-Tween 20 buffer, the membrane was incubated with a casein-block diluted (1:3,000) secondary antibody against goat IgG for 1 hour at room temperature. Membranes were transferred to shallow trays and incubated with 1-step Ultra TMB according to the manufacturer's protocol. The color development process was carefully monitored and briefly washed in water when a band of desired intensity was achieved.

Purified Aqueous Two-Phase Extraction of rhIL-11 Soluble but misfolded rhIL-11 was precipitated from the fermentation medium by aqueous two-phase extraction. Solid PEG 8000 was added directly to the fermentation medium filtrate to give a final concentration of 6%-8% (w/v). The solid polymer was completely dissolved by gentle agitation, followed by centrifugation at 4,000 rpm for 10 minutes to collect the precipitated protein.

Refolding of rhIL-11 The precipitated protein was dissolved in 20 mM sodium phosphate containing 7 M guanidine hydrochloride (GdHCl) pH 8-9 buffer to a final concentration of 2 mg/mL and incubated for 1 hour at room temperature. Denatured proteins were properly refolded by adding 11 volumes of 4 mM sodium phosphate buffer (pH 8) to dilute the GdHCl and incubating the solution for 2 hours at room temperature. Prior to ion exchange chromatography, the resulting solution was diluted with 4 mM phosphate buffer by simple dilution or buffer exchange using ultrafiltration or dialysis to give a conductivity of less than 6.5 mS/cm. got

Cation Exchange Chromatography Ion exchange chromatography was performed using commercially available chromatography equipment (eg AKTAprime plus from GE Healthcare Life Sciences). The resulting dilutions were centrifuged or filtered through 0.45 or 0.2 μm membranes to remove particulates prior to loading onto the chromatography column. The mixture was loaded onto a Capto S column equilibrated with buffer A containing 20 mM sodium phosphate pH8. Eluted with a gradient or step elution of buffer B containing 20 mM sodium phosphate pH 8 and 1 M NaCl.

Hydrophobic Interaction Chromatography Hydrophobic interaction chromatography was performed using commercially available chromatography equipment (eg, AKTAprime plus from GE Healthcare Life Sciences). Fractions containing rhIL-11 from the cation exchanger (CaptoS) were pooled and added to 0.5 M ammonium sulfate containing 5 mM DL-methionine. The resulting dilutions were centrifuged or filtered through 0.45 or 0.2 μm membranes to remove particulates prior to sample loading. The mixture was loaded onto a Butyl HP column equilibrated with buffer A containing 0.5 M ammonium sulfate and 5 mM DL-methionine in 10 mM sodium phosphate pH 7 buffer. Proteins were eluted with a step or gradient elution of buffer B containing 10 mM sodium phosphate pH 7 buffer. Additional 0.2 M acetic acid was used to elute strongly bound rhIL-11.

Purity as Determined by Size Exclusion Chromatography The content of covalently and non-covalently aggregated rhIL-11, in addition to other high molecular weight species, was determined using a commercial UPLC system equipped with a diode array detector (eg, It was analyzed by size exclusion chromatography (SEC) using an UltiMate 3000 Rapid Separation LC system from Thermo Scientific). Chromatographic procedures were performed using:

Column: Waters Acquity BEH200 SEC 1.7 μm, 4.6×150 mm, 300 A (Angstroms) pore size (part number 186005225), equipped with a guard cartridge (part number 186005793) Mobile phase: 25 mM sodium phosphate containing 0.5 M NaCl pH 7.0
Flow rate: 0.3 ml/min Detection: 280 nm
Stop time: 10 min Injection of 5 μg protein

Purity and Molecular Weight Determined by LC/MS The purity of rhIL-11 was determined by (a) UPLC (e.g. UltiMate 3000 from Thermo Scientific) equipped with a diode array detector connected to an Agilent 6540 UHD Accurate Mass Q-TOFLC/MS system. Analysis was by reverse phase (RP) chromatography using a Rapid Separation LC system, or an Agilent 1290 infinity UPLC system). The chromatographic procedure was performed using the following procedure.

Columns: ACQUITY UPLC PST C18 column, 300A (Angstrom) pore size, 1.7 μm, 2.1 mm×150 mm (part number 186003687), Acquity BEH C18 VanGuard Pre-column, 300A (Angstrom) pore size, 1.7 μm, 2.1 ×5mm (part number 186004629)
Mobile phase and gradient: Mobile phase A: 0.1% (v/v) trifluoroacetic acid (TFA) in water; Mobile phase B: 0.1% (v/v) TFA in 95% (v/v) acetonitrile.
Flow rate: 0.4 ml/min Column temperature: Ambient temperature Detection: 214 nm
Injection volume: 5 μg

Gradients—A typical solvent gradient is shown in Table 2.

The operating parameters for mass spectrometry were as follows.
m/z range and polarity: 150-3000 positive Source parameters: gas temperature 300° C.; gas flow 8 L/min Nebulizer 35 psig
Sheath gas temperature 380° C.; sheath gas flow rate 11 L/min Scan source parameters:
V Cap = 3500
Nozzle voltage 1,000V
Fragmentor 175
Skimmer 65
Octopole RF Peak 750

Cell-Based Bioassays The biological activity of rhIL-11 was calculated in cell proliferation assays using the 7TD1 cell line. IL-11 reference standards and unknown samples were diluted aseptically to give a concentration range of 10-fold from 20,000 ng/ml to 0.2 pg/ml (9 dilutions total). 50 μL of rhIL-11 standard or sample was added twice to wells containing 7TD1 cells at 4,000 cells per well (eg, wells of a 96-well plate). Cells were incubated at 37° C. in a humidified atmosphere containing 5% CO ₂ to characterize their response to different IL-11 concentrations for 3 days in the presence of 2 μg/mL IL-11 receptor ( 12). For the cell proliferation assay, 20 μL of MTS solution was dispensed into wells using a multichannel pipette and incubated for 2.5-3 hours in a 37° C. incubator depending on signaling. After incubation, plates were read for absorbance at 490 nm using a UV/Vis microplate and cuvette spectrophotometer (eg Multiskan GO from Thermo Scientific).

A dose-response curve was generated by plotting absorbance at 490 nm on the y-axis against concentration on the x-axis by fitting a sigmoidal dose-response curve to a four-parameter nonlinear logistic equation using GraphPad software Prism6. EC50 (half maximal effective concentration) of
y=((ad)/(l+(x/c)b))+d
here,
a is the y-axis of the minimum asymptote as the concentration approaches zero; b is the gradient representing the gradient of the curve c is the _EC50 d is the maximum asymptote as the concentration approaches infinity is the y-axis of x is the concentration y is the absorbance at 490 nm Specific bioactivity is derived from the following equation.
Specific activity (U/mg) = (reference specific activity) x (EC ₅₀ reference/EC ₅₀ sample)

Secondary Structure Characterization by Circular Dichroism Far-UV circular dichroism (CD) spectra were recorded at room temperature using a Jasco J-815 spectropolarimeter with a quartz cell of 1.0 cm path length. Protein samples were diluted to approximately 0.02 mg/mL with deionized water or 5 mM sodium phosphate (pH 7). Secondary structure was monitored in the deep UV region (190 nm-250 nm) with operating parameters including scan speed, bandwidth and response set at 100 nm/min, 1 nm and 2 seconds, respectively. Spectra were obtained from the average of three scans.

Peptide Mapping Proteolysis solutions were prepared in 50 mM sodium phosphate pH 8 buffer containing 2 mg/mL protein by adding sequencing grade 1/100 (w/w) trypsin. After incubation for 6 hours at room temperature, TFA (or formic acid) was introduced to a final concentration of 0.1% to stop the reaction. Precipitates were removed by centrifugation or filtration through 0.2 or 0.4 μm membranes prior to injection. Peptide identification was performed using an LC/MS system—Agilent 1290 Infinite UPLC system combined with an Agilent 6540 UHD Accurate Mass Q-TOF LC/MS system. The chromatographic procedure was performed as follows.

Column: Zorbax 300 SB-C8, 2.1 x 150 mm, 5 μm, 300 A (Angstrom) pore size (Agilent part number 883750-906)
Mobile phase and gradient: Mobile phase A: 0.1% (v/v) TFA in water; Mobile phase B: 0.1% (v/v) TFA in 95% (v/v) acetonitrile
Flow rate: 0.2 ml/min Detection: 214 nm
Injection volume: 10 μg

Gradients—A typical solvent gradient is shown in Table 3.

The operating parameters for mass spectrometry were as follows.
m/z range and polarity: 400-1700 positive Source parameters:
Gas temperature 300℃
Gas flow 8 L/min Nebulizer 35 psig
Sheath gas temperature 350°C
Sheath gas flow rate 11 L/min Scan source parameters:
V Cap = 3500
Nozzle voltage 1,000V
Fragmentor 175
Skimmer 165

衝突エネルギー

collision energy

The estimated molecular weights of proteolytic peptides cleaved by trypsin are listed in Table 1. Identification of each proteolytic peptide was performed manually using the MS Product tool, which generates fragment ions that can arise from peptide fragmentation in the m/z spectrum. Tryptic peptides obtained from recombinant IL-11 are shown in Table 5.

Amino Acid Composition Amino acid composition was determined using a Hitachi High-Speed Amino acid Analyzer L-8900. This provides for separation of hydrolyzed amino acids by an ion exchange mechanism followed by derivatization with ninhydrin. Prior to hydrolysis, protein samples were prepared at 1 mg/mL in triplicate and hydrolyzed in 6N HCl at 110° C. for 22 hours under nitrogen atmosphere. After evaporation in a water bath at 80° C., the hydrolyzed samples were resuspended in 0.02N HCl. Under such acidic hydrolysis conditions, asparagine and glutamine are deamidated to form their respective acids. Tryptophan is completely degraded. Cysteine and methionine are oxidized and not readily detected from acid hydrolysates. Tyrosine, serine and threonine are partially hydrolyzed (13).

High Cell Density Fermentation A glycerol-fed batch fermentation was performed in a 1-L fermentor inoculated with a protease-deficient strain (pPINKS1-IL11-24) thawed from a vial of a research working cell bank for downstream purification runs. A typical cell growth curve is shown in FIG. 1A. FIG. 1A shows cell cultures that achieved 200 OD after additional feeding of glycerol. Cell density continued to grow up to 250 OD after methanol induction, but gradually declined after 22 hours post-induction, probably due to insufficient nutrients. Fermentation broth samples were taken at 22, 46, and 70 hours after induction and analyzed by non-reducing SDS-PAGE. Figures 1B and 1C show typical results of SDS-PAGE and immunoblotting, respectively. Expression levels were not determined by ELISA, as this method was found to consistently underestimate productivity. Instead, rhIL-11 productivity was estimated by the intensity of Coomassie blue staining compared to that of a reference standard, either by visual inspection or densitometry, and measured approximately 0.4-0.6 g/L. was found to occur. SDS-PAGE performed by Coomassie blue staining showed several distinct bands below the position of rhIL-11 (approximately 20 KD) at approximately 15 KD (FIG. 1B), which was confirmed by immunoblotting with an antibody against rhIL-11. It was a degraded species as verified (Fig. 1C). The expression productivity of the fermentation medium determined by RP-HPLC was approximately 0.4 mg/mL.

Capture of IL-11 Using Cation Exchange Chromatography Given the high isoelectric point of IL-11 (pI=11), cation exchange chromatography was attempted to capture rhIL-11 from the fermentation medium at pH8. After removing the cell paste by centrifugation, hundreds of milliliters of broth were subjected to buffer exchange by ultrafiltration or directly diluted with at least 10 volumes of water to obtain a conductivity of less than 3 mS/cm. To recover rhIL-11, a small volume of solution corresponding to approximately 2 milligrams of rhIL-11 was loaded onto a 1 mL Capto-S column (cation exchanger) pre-equilibrated with 20 mM Tris pH 8 buffer. Bound proteins were eluted with a NaCl gradient up to 1M NaCl. SDS-PAGE and immunoblotting results showed that rhIL-11 was found in the flow-through fraction and did not bind to the column (data not shown). To ensure suitability of the purification system using cation exchangers, 0.5 mg of reference rhIL-11 was spiked into 1 mL fermentation broth as a positive control, followed by the purification procedure described above. A recovery of approximately 30% was successfully eluted by a NaCl gradient in the same manner, suggesting the suitability of the cation exchanger for isolating rhIL-11. Isolation using an anion exchanger (Capto-Q) was attempted by collection of the flow-through material. A small volume of solution corresponding to approximately 2 milligrams of rhIL-11 was loaded onto a 1 mL Capto-Q column (anion exchanger) and rhIL-11 was recovered in the flow-through. The column was pre-equilibrated with 20 mM Tris pH 8 buffer and then eluted with a NaCl gradient up to 1 M NaCl. Surprisingly, SDS-PAGE results showed successful recovery of rhIL-11 in the NaCl-containing fractions, but not in the flow-through (data not shown). This purification process was repeated leading to consistent and reproducible results, confirming the unexpected ionic properties of the expressed rhIL-11.

We noted the weak bioactivity of rhIL-11 in cell-based proliferation assays and the underestimation of rhIL-11 content using ELISA quantification when using samples of fermentation media. Interestingly, bioactivity was restored when the fermentation broth was overdosed with reference rhIL-11 (data not shown). We conclude that unexpected binding (or lack thereof) to ion exchangers and loss of biological activity indicate changes in the physical properties of the expressed protein.

Liquid Two-Phase Extraction Prior to further investigation, the expressed protein was recovered from the fermentation medium by a simple aqueous two-phase extraction. The extraction was developed to precipitate the non-native rhIL-11 from the liquid phase by adding polyethylene glycol 8000. Solid PEG-8000 was added to 1 mL of filtered fermentation broth to yield 2.9, 3.8, 5.0, 6.2, 7.3 and 8.2% (w/v) respectively. After 1 hour incubation at 4° C., turbidity was visible only in samples containing 6.2, 7.3 and 8.2% PEG8000. The precipitate was collected by centrifugation for 5 minutes at maximum speed in a tabletop centrifuge and the precipitated protein was reconstituted with 1 mL pH 8 sodium phosphate buffer. As shown in Figure 2, almost all rhIL-11 could be recovered from the precipitate by adding as little as 6% (w/v) PEG8000. In subsequent studies rhIL-11 was recovered from the culture medium by precipitation with PEG unless otherwise stated.

Structural Characterization of Secreted rhIL-11 in Fermentation Media Structural characteristics were studied using circular dichroism (CD) and size exclusion chromatography. Precipitated rhIL-11 recovered from liquid two-phase extraction by adding 8% PEG8000 to the fermentation medium was resuspended in deionized water at approximately 0.02 mg/mL. A typical CD spectrum is shown in FIG. This reveals a significant helical structure (as indicated by negative bands at about 222 nm and 210 nm and a positive band at about 195 nm). This suggests that crude rhIL-11 maintained a helical backbone.

For size exclusion studies, PEG-precipitated proteins were resuspended in 20 mM sodium phosphate pH 7 buffer to a concentration of approximately 1 mg/mL. Molecular size was compared to a reference standard of rhIL-11 using an Acquity BEH200 SEC column capable of separating globular proteins ranging from 10K to 450KDa. The results, as seen in Figure 4, demonstrate that crude rhIL-11 from the fermentation medium has an unexpectedly high molecular weight. This suggests that secreted rhIL-11 exists as soluble aggregates in the culture broth. Combined with the CD data, it appears that although the helical structure of secreted rhIL-11 is maintained, the monomeric molecules tend to non-covalently associate with each other to form intermolecular helical bundles. The inventors, without being bound by theory, believe that this is the cause of the low yields in the original process, resulting in altered physicochemical properties and loss of biological activity.

Prevention of self-aggregation using nonionic detergents in fermentation media Pichia pastoris expresses proteins via the eukaryotic folding pathway, but self-aggregation and misfolded secretion in high-density yeast cultures Type rhIL-11 has not been previously reported. Aggregated forms cause significant problems such as loss of biological activity and low yields in downstream purification steps. One strategy to prevent flocculation is to introduce additives or surfactants into the fermentation medium (14). To investigate this, 0.1% Tween-80 was introduced into 1 L of high-density fed-batch fermentation medium. After harvesting, 10x water was added to 130 mL of fermentation medium to lower the conductivity. After removing particulate matter by simple filtration through a 0.45 or 0.2 μm membrane, the resulting solution was loaded onto a cation exchanger (Capto-S) to recover bioactive rhIL-11. A typical purification profile is shown in FIG. After loading the column, the column was washed with a 50 mM NaCl buffer followed by a 50-300 mM NaCl gradient over 10 column volumes. Fractions containing rhIL-11 were combined as determined by SDS-PAGE analysis of selected fractions to give a step yield of 13.6%. The findings suggested that 0.1% Tween-80 could reduce self-aggregation during fermentation to some extent, but the recovery of bioactive rhIL-11 may not be satisfactory.

Optimization of Refolding for rhIL-11 Restoration Protein refolding in solution is the result of many physicochemical factors, including ionic strength, pH, temperature, protein concentration, and the like. Urea, ammonium sulfate, SDS and guanidine hydrochloride (GdHCl) were investigated as denaturants that may be useful in the refolding process of rhIL-11. Proteins were precipitated from 2 mL of fermentation medium after PEG precipitation and reconstituted to 5 mg/mL with respective denaturant-containing buffers: 8 M urea, 1 M ammonium sulfate, 0.5% SDS or 6 M GdHCl. After dilution with water to achieve a conductivity of less than 5 mS/cm, only native and biologically active rhIL-11 is expected to be adsorbed to the cation exchanger and eluted by the NaCl salt gradient. The results showed that, with the exception of 6M GdHCl, denaturants failed to refold rhIL-11, as shown in FIG. 11 was successfully eluted using a NaCl gradient). In other studies, proteins precipitated from 2 mL of fermentation medium after PEG precipitation were 2 mL of alkaline solutions consisting of 0.1 N NaOH, 0.1 N NaOH with 70% ethanol, or 0.1 N NaOH with 1% Tween 20. dissolved in After dilution with water and adjustment of the pH to pH 8, cation exchangers recovered little native rhIL-11 from such solutions. The results suggest that rhIL-11 can be successfully renatured in high yield using guanidine hydrochloride in a preferred embodiment of the present concept.

The protein concentration used in the refolding process was optimized by dissolving PEG protein precipitates at various protein concentrations in 7M GdHCl solution. Proteins from 2 mL of fermentation medium were collected after PEG precipitation and reconstituted at various protein concentrations in 7 M GdHCl buffer solution. After refolding by simple dilution, the refolded rhIL-11 of each preparation was isolated on a cation exchanger as described above. Refolding yields were assessed by comparing the observed peak areas with that of the rhIL-11 reference standard, as shown in FIG. The results show that refolding occurred at all concentrations tested, with optimal broad yields at a protein concentration of approximately 2 mg/mL.

A number of co-solutes that act as folding promoters or aggregation inhibitors can be introduced to aid in protein refolding. Typical co-solutes include PEG, cyclodextrin, arginine, proline, and sucrose. The mechanism of action for these co-solutes is unclear. We studied the effect of two co-solutes, 0.5M arginine and 0.4M sucrose, added to the refolding solution. After refolding by dilution, the refolded rhIL-11 of each preparation was isolated on a cation exchanger as described above. Refolding yields were assessed by evaluating peak heights (typical results are shown in FIG. 8), indicating similar yields of refolded rhIL-11. We have found that neither arginine nor sucrose promote refolding of rhIL-11 and that the use of co-solutes can be eliminated from the rhIL-11 purification process. This advantageously simplifies downstream purification steps.

The pH for refolding was optimized by dissolving protein precipitates generated by PEG treatment at various pHs. Protein from 2 mL of fermentation medium was recovered after reconstitution of the PEG precipitate to 2 mg/mL in 7 M GdHCl buffers at different pH values. After refolding by simple dilution with the corresponding pH solution, the refolded rhIL-11 of each preparation was isolated on a cation exchanger using the procedure described above. Refolding yields were assessed by comparing the observed peak areas to those produced by rhIL-11 reference standards, as shown in FIG. Refolding was seen at all pH values studied, with a broad optimum around pH 8.5.

Isolation of Refolded rhIL-11 Using Cation Exchange Chromatography Isolation using cation exchange medium was investigated using 236 mL of fermentation medium containing approximately 94.2 mg of secreted rhIL-11. Proteins were precipitated with 8% PEG8000 (w/v) and collected by centrifugation at 4,000 rpm for 15 minutes. After discarding the supernatant, the solid phase was reconstituted to a 2 mg/mL protein concentration with 20 mM sodium phosphate pH 8 buffer containing 7 M GdHCl. The protein solution was incubated at room temperature for 1 hour with gentle agitation to allow complete solubilization. The renaturation/refolding process was initiated by pouring the protein solution into 10 volumes of 4 mM sodium phosphate pH 8 buffer without guanidine. The resulting solution was further incubated at room temperature for an additional hour and then either buffer exchanged using ultrafiltration or diluted directly with additional buffer to achieve a conductivity of less than 6 mS/cm. Cation exchange chromatography was performed using a Capto-S column (1 cm×10 cm) and typical results are shown in FIG. After loading the column with a sample load of 12 mg/mL resin, the column was washed with 50 mM NaCl buffer followed by application of a 50-300 mM NaCl gradient over 10 column volumes. Fractions containing rhIL-11 (as shown by non-reducing SDS-PAGE) were combined in pools to give step yields of about 40% to about 60%. The products were also analyzed by RP-UPLC coupled with mass spectrometry to elucidate the molecular weight of the purified protein (see Figure 11). The deconvoluted mass of the main peak observed by UPLC was 19046.7 Da, consistent with the theoretical average mass of rhIL-11 of 19,047 Da. A small peak (accounting for 8.6%) ahead of rhIL-11 in the liquid chromatogram is oxidized because its deconvoluted mass is 15.8 Da greater than that of rhIL-11, which corresponds to the mass of approximately a single oxygen. thought to represent a species (16 Da). Purified rhIL-11 after the refolding process was subsequently subjected to cell-based assays, suggesting full recovery of activity by the refolding process (data not shown).

A small shoulder eluting after the dominant peak from the Capto-S column was further investigated. RP-UPLC analysis coupled with MS showed a single peak with the same retention time as the rhIL-11 reference standard and a molecular weight consistent with its theoretical mass (data not shown). SDS-PAGE analysis also revealed a migration position identical to the rhIL-11 reference standard (data not shown). On size exclusion chromatography using 25 mM sodium phosphate pH 7.0 containing 0.5 M NaCl as the eluent, this small shoulder eluted as a doublet rather than a single peak. One peak of the doublet eluted at the same retention time as native rhIL-11, the remaining peak eluted earlier and is thought to be in the dimeric form (see Figure 12). These fractions were combined with the main pool as subsequent purification using hydrophobic interaction chromatography with 0.5 M ammonium sulfate could potentially disrupt such aggregates.

Reduction of oxidized rhIL-11 by adding peptones of non-animal origin to the fermentation medium Protein oxidation results from constant exposure to various forms of reactive oxygen species (ROS) such as oxygen radicals during aerobic fermentation. , a very common source of product-related impurities during the manufacturing process (performed using basal medium and 0.9% ammonium sulfate as nitrogen source). After purification from the Capto-S column, the oxidized rhIL-11 content of the resulting purified rhIL-11 was approximately 12.6% as determined by RP-UPLC. Ammonium sulfate was replaced with 0.5% phytone peptone to reduce the content of oxidation products. Phytone peptone is derived from soy peptone, which contains sulfur-containing amino acids that can not only provide a nitrogen source, but also eliminate or reduce the formation of ROS during fermentation. The inventors believe that supplementing the fermentation medium with other sulfur-containing peptides, amino acids (eg, cysteine, methionine), and/or organic compounds will have similar effects. After purification on the Capto-S column, the amount of oxidized rhIL-11 obtained in the modified fermentation medium was reduced to approximately 6.6%, close to the proposed acceptance criteria of 5.0%. The product was further polished by a second chromatography step to remove oxidized rhIL-11 using hydrophobic interaction chromatography.

Removal of Oxidized rhIL-11 Using Hydrophobic Interaction Chromatography To the combined pool of fractions eluted from the Capto-S column, solid DL-methionine was introduced to achieve a concentration of approximately 5 mM and approximately 0. Solid ammonium sulfate was added to obtain a concentration of 0.5M. After filtration through a 0.2 or 0.45 μm membrane, the resulting protein solution was loaded onto a Butyl HP column (1 cm×10 cm) with a sample load of 8 mg/mL total volume followed by 0.5 M ammonium sulfate. Washed with buffer solution. The column was then washed with over 15 column volumes of buffer containing 0.475 M ammonium sulfate. The polished rhIL-11 product was eluted with 10 mM sodium phosphate pH 7 buffer (see Figure 13). The oxidized rhIL-11 content of each fraction was quantified by RP-UPLC, showing a decrease in the content of oxidized species. Fractions containing less than 5% oxidized species were pooled to give a process recovery of approximately 49%.

Buffer Changes and Concentrations The product eluted from the Butyl HIC column contained large amounts of ammonium sulfate, which was subsequently removed by extensive dialysis and/or ultrafiltration against 10 mM sodium phosphate pH 7 buffer. . The concentration was adjusted to 6 mg/mL or higher for refrigerated storage.

Characterization of the rhIL-11 Product The final purified bulk rhIL-11 was subjected to various analyzes including identity, purity and potency. Purity and relative molecular weight were analyzed by non-reducing SDS-PAGE using a 16% gel (as shown in Figure 14), which showed a single band with high purity.

Purity (and related impurities) was determined by RP-UPLC. As shown in Figure 15, the purity of rhIL-11 was greater than 95%. The rhIL-11 preparation contains approximately 2.5% oxidized rhIL-11, which is comparable to the 2.4% content of the commercial product (Nemega). Monomeric rhIL-11 content was quantified by SEC-UPLC as shown in FIG. 16 and the purity was 99%.

The molecular weight of purified rhIL-11 was determined by LC-MS and showed a deconvoluted mass at 19,045.7 Da (see Figure 17). This is in good agreement with the theoretical average molecular weight of 19,047 Da (deviation -68.2 ppm). Additionally, the amino acid sequence of purified rhIL-11 was characterized by peptide mapping coupled with LC-MS. Proteins were digested by trypsin. Trypsin selectively hydrolyzed peptide bonds C-terminal to lysine and arginine amino acid residues. Identity of each peak was determined by m/z ratio and MS/MS fragmentation. All proteolytic peptides except T11, T16 and T19 were successfully assigned (as shown in Figure 18), providing 88.7% sequence coverage.

The N-terminal amino acid sequence was determined using the Applied Biosystems LC494 Procise® Protein Sequencing System and the first 15 amino acids were confirmed as follows. Gly-Pro-Pro-Pro-Gly-Pro-Pro-Arg-Val-Ser-Pro-Asp-Pro-Arg-Ala. Hydrolyzed amino acid composition was determined after sample pretreatment with 6N HCl hydrolysis at 110° C. for 22 hours under nitrogen. Under such acidic hydrolysis conditions, asparagine and glutamine are deamidated to form their respective acids. Tryptophan is completely degraded. Cysteine and methionine are oxidized and not readily detected from acid hydrolysates. Tyrosine, serine and threonine are partially hydrolyzed. The results of amino acid composition studies are shown in Table 6. The molar ratios of most amino acids are in good agreement with the expected values. We believe that the results observed for isoleucine, tyrosine and phenylalanine were influenced by their relatively low abundance.

Ｎ／Ａ：該当しない。
ＢＤ：検出限界以下。最適濃度範囲は０．４から１０nmole／20μL。

N/A: Not applicable.
BD: below detection limit. The optimal concentration range is 0.4 to 10 nmole/20 μL.

Biological activity of purified rhIL-11 was determined using the 7TD1 cell proliferation assay. Typical results are shown in FIG. The observed EC50 was 1.1 and 2.2 ng/mL for reference rhIL-11 and rhIL-11 produced by the method described, respectively, suggesting comparable potency.

As indicated above, the concepts of the present invention provide novel methods for the production and isolation of highly pure rhIL-11 using yeast. Secreted recombinant human interleukin-11 was successfully expressed by Pichia pastoris, but the expression product was biologically inactive due to self-aggregation of rhIL-11. Addition of non-ionic detergents such as Tween-80 in high-density fed-batch cultures resulted in only about 10% bioactive products of total rhIL-11. However, addition of Tween-80 may result in foaming due to agitation during the fermentation process.

It should be apparent to those skilled in the art that many more modifications than those already described are possible without departing from the inventive concept herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest manner consistent with the context. In particular, the terms "comprising" and "comprising" are to be interpreted as referring non-exclusively to an element, component or step, which means that the referenced element, component or A step is meant to be present, utilized, or may be combined with other elements, components, or steps not explicitly referenced. When a claim in the specification refers to at least one selected from the group consisting of A, B, C... and N, the text need only one element from that group, not A+N or B+N. should be interpreted as

References
1. Du XX, Williams DA. Interleukin-11: A Multifunctional Growth Factor Derived from the Hematopoietic Microenvironment. Blood. 1994;83(8):2023-30.
2. Unattributed Editorial; Transgenic Milk Prospects Turn Sour. Nat Biotech. 2006;24(4):368.
3. McCoy J, DiBlasio-Smith E, Grant K, Vallie EL, inventors; Genetics Institute, assignee. Peptide and Protein Fusions to Thioredoxin, Thioredoxin-like Molecules and Modified Thioredoxin-like Molecules. US patent 5,646,016.
4. Jung Y, Ho SH, Park MO, Kim MS, inventors Biopolymer Conjugates Comprising an Interleukin-11 Analog. USA patent application 2010/0098658.
5. Cox-III G, inventor; Bolder Biotechnology, Inc., assignee. Cysteine Variants of Interleukin-11. US patent 8,133,480.
6. Wang TY, Wang CM, Wei G, Qiu JW, Huang YS. Expression of the Recombinant Human Interleukin-11 in Pichia pastoris. Acta Biochimica et Biophysica Sinica. 2001;33(6):659-64.
7. Qiu J, Wang T, Liu GA, inventors; Jiuyuan Gene Engineering Co. Ltd., assignee. Method for Producing Recombinant Human Interleukin- 11 using Methyl Alcohol Yeast. China patent 1288062A. 2001.
8. Huang Y, Ma K, Yang T, Sun H, Shu F, Chou J, inventors; Jiuyuan Gene Engineering Co. Ltd., assignee. Production method for Pichia pastoris expression recombinant human interleukin 11. Chinese patent 102329388. 2013.
9. Hong E, Davidson AR, Kaiser CA. A Pathway for Targeting Soluble Misfolded Proteins to the Yeast Vacuole. The Journal of cell biology. 1996;135(3):623-33.
10. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins M, Appel R, et al. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, editor. The Proteomics Protocols Handbook. USA: Humana Press Inc 2005. p.571-607.
11. Hart RA, Lester PM, Reifsnyder DH, Ogez JR, Builder SE. Large Scale, in situ Isolation of Periplasmic IGF-I from E. coli. Bio/Technology (Nature Publishing Company). 1994;12(11): 1113 -7.
12. Karow J, Hudson KR, Hall MA, Vernallis AB, Taylor JA, Gossler A, et al. Mediation of Interleukin- 11 -Dependent Biological Responses by a Soluble Form of the Interleukin- 11 Receptor. The Biochemical journal. 1996;318 (Pt 2): 489-95.
13. Ozols J. Amino acid analysis. Methods in Enzymology. 1990;182:587-601.
14. Bahrami A, Shojaosadati SA, Khalilzadeh R, Mohammadian J, Farahani EV, Masoumian MR. Prevention of Human Granulocyte Colony-Stimulating Factor Protein Aggregation in Recombinant Pichia pastoris Fed-Batch Fermentation using Additives. Pt 2): 141-8.
15. Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical Considerations in Refolding Proteins from Inclusion Bodies. Protein Epression and Purification. 2003;28(l):l-8.
16. Yokota H, Saito H, Masuoka K, Kaniwa H, Shibanuma T. Reversed Phase HPLC of Met58 Oxidized rhIL-11: Oxidation Enhanced by Plastic Tubes. Journal of Pharmaceutical and Biomedical Analysis. 2000;24(2):317-24 .
17. Farr SB, Kogoma T. Oxidative Stress Responses in Escherichia coli and Salmonella typhimurium. Microbiological Reviews. 1991;55(4):561-85.

Shacter E. Quantification and Significance of Protein Oxidation in Biological Samples. Drug Metabolism Reviews. 2000;32(3-4):307-26

Claims (19)

introducing into yeast an expression vector encoding recombinant IL-11, wherein said recombinant IL-11 is not in the form of a fusion protein.
culturing the yeast in a medium under conditions that induce the expression of IL-11;
separating the supernatant from the solids of said medium;
identifying soluble but misfolded recombinant IL-11 in said supernatant;
contacting the supernatant with polyethylene glycol in an amount sufficient to form a suspension containing the precipitate;
solubilizing the precipitate in a solution containing a denaturant to produce a crude IL-11 solution;
reducing the concentration of the denaturant to 0.7 M or less to produce a refolded IL-11 solution;
contacting the refolded IL-11 solution with an ion exchange medium; and
eluting purified IL-11 from said ion exchange medium. The method of claim 1, wherein said polyethylene glycol is provided at a final concentration of 4 % (w/v) to 12 % (w/v). 3. The method of claim 1 or 2, wherein said polyethylene glycol has an average molecular weight of 2,000D to 20,000D . A method according to any one of claims 1 to 3, wherein said denaturant is selected from the group consisting of urea, guanidine hydrochloride and surfactants. 5. The method of claim 4, wherein said surfactant is selected from the group consisting of dodecyl sulfate and N-sarkosyl. The method according to any one of claims 1 to 5, wherein in the solubilizing step, the denaturant is guanidine hydrochloride at a concentration of 4 M to 10 M. The method of any one of claims 1 to 6, wherein the step of reducing the concentration of the denaturant comprises incubating at 18°C to 25°C for 1 hour after reducing the concentration of the denaturant. . The method of any one of claims 1-7, wherein the ion exchange medium comprises a cation exchange medium. 9. The method of any one of claims 1-8, wherein the step of reducing the concentration of the denaturant is achieved by dilution of the crude IL-11 solution. The method of any one of claims 1-9, wherein the step of reducing the concentration of the denaturant is achieved by buffer exchange of the crude IL-11 solution. The step of producing the refolded IL-11 solution comprises: 0 . A method according to any one of claims 1 to 10, which is carried out at a protein concentration of 1 mg/ml to 10 mg/ml. A method according to any one of claims 1 to 11,
Further steps of
contacting the purified IL-11 with a hydrophobic interaction medium; and
eluting polished IL-11 from the hydrophobic interaction medium;
wherein said polished IL-11 has a reduced content of oxidized IL-11 compared to said purified IL-11. 13. The method of claim 12, wherein said polished IL-11 has a purity of at least 95 %. 13. The method of claim 12, wherein said polished IL-11 contains 5 % or less oxidized IL-11. 13. The method of claim 12, wherein the polished IL-11 contains no more than 1 % dimers of IL-11. 16. The method of any one of claims 1-15, wherein the step of producing the refolded IL-11 solution is performed in the absence of a co-solute. 17. The method of any one of claims 1-16, wherein the step of producing the refolded IL-11 solution is performed at a pH of 4-12 . 18. The method of any one of claims 1-17, wherein the step of producing the refolded IL-11 solution is performed at a pH of 7-11 . When tested with the 7TD1 cell line, polished IL-11 was 4 ×10 ⁶ U/mg˜1 . A method according to any one of claims 1 to 18, having a biological activity of 2 x ¹⁰⁷ U/mg.

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